Significance of quantitative analyses of the impact of heterogeneity in mitochondrial content and shape on cell differentiation

Mitochondria, classically known as the powerhouse of cells, are unique double membrane-bound multifaceted organelles carrying a genome. Mitochondrial content varies between cell types and precisely doubles within cells during each proliferating cycle. Mitochondrial content also increases to a variable degree during cell differentiation triggered after exit from the proliferating cycle. The mitochondrial content is primarily maintained by the regulation of mitochondrial biogenesis, while damaged mitochondria are eliminated from the cells by mitophagy. In any cell with a given mitochondrial content, the steady-state mitochondrial number and shape are determined by a balance between mitochondrial fission and fusion processes. The increase in mitochondrial content and alteration in mitochondrial fission and fusion are causatively linked with the process of differentiation. Here, we critically review the quantitative aspects in the detection methods of mitochondrial content and shape. Thereafter, we quantitatively link these mitochondrial properties in differentiating cells and highlight the implications of such quantitative link on stem cell functionality. Finally, we discuss an example of cell size regulation predicted from quantitative analysis of mitochondrial shape and content. To highlight the significance of quantitative analyses of these mitochondrial properties, we propose three independent rationale based hypotheses and the relevant experimental designs to test them.


Introduction
Over the course of evolution, the prokaryote-turned-cellular-organelles, mitochondria, have been integrated into the functionality of the majority of the eukaryotic cells, almost in a customized way.Today we know mitochondria as multifunctional organelles with marked heterogeneity at different levels, which has been covered extensively in the reviews cited below in this and the following sections.Quantitative knowledge of the heterogeneity of various mitochondrial properties is necessary for understanding the critical nuances of mitochondrial functions and dysfunctions.In this review, we provide a necessary overview of the organelle and critically evaluate the current understanding of the quantitative aspects of certain mitochondrial properties in a certain cellular process towards generating multiple independent hypotheses.
Mitochondrial content varies between tissue and cell types, with differentiated aerobic cells at the maximum end and stem cells (undifferentiated) at the minimum end of the spectrum [1,2].Mitochondria are also morphologically and structurally distinct between and within various tissue/cell types, and are also remodelled by various stimuli [3][4][5][6].Within the same tissue type, mitochondrial shape and structure can vary based on local signalling [7][8][9].Cells like neurons and myocytes exemplify striking intracellular heterogeneity of mitochondrial shape and function [10,11], while the same has been more recently documented for adipocytes [12].Mitochondrial heterogeneity also exists between individual human subjects at the level of the circular DNA they carry (mt-DNA), which contributes to genetic variation [13].Other than the classically described function of ATP production, mitochondria play critical roles in metabolic, redox and calcium homeostasis, which vary depending on the tissue type [5,14].Mitochondria also serve as a pivot point of decision-making in the cell death process by controlling apoptosis [15].Research over past decades has also revealed mitochondria as signalling organelles [16,17].Therefore, a cell needs to maintain the right content, shape and kind of mitochondria that is achieved by regulating a mitochondrial life cycle.In such a life cycle, the mitochondrial content is controlled by the balance of mitochondrial biogenesis and mitophagy (mitochondrial clearance), while mitochondrial shape is controlled by a balance of mitochondrial fission and fusion processes [18].
Multifaceted involvement of mitochondria in various developmental and regenerative processes has gained wider attention.During normal development, pluripotent embryonic stem cells proliferate and differentiate into embryonic cell lineages that further differentiate into quiescent cells of various tissues [19].Some of the differentiated cells in our adult body proliferate further to play critical roles in wound healing, immunity and integrity of various organs.Moreover, various adult tissues harbour adult stem cells that self-renew, proliferate and differentiate to replenish lineage specific dying cells.A selfrenewing or proliferating cell has to exit the proliferating cycle to enter differentiation, which has to be regulated in a timely fashion.Aberrations in cell proliferation and differentiation can lead to various developmental, neurodegenerative and age-related disorders as well as cancer.Therefore, it is key to understand the fundamental concepts of mitochondrial involvement in cell proliferation and differentiation of stem cells and their progenies.
Here, we focus on two properties of mitochondria, namely mitochondrial content and shape, to highlight the significance of quantitative analyses of mitochondria.First, we critically review and evaluate the advances in the quantitative measurement of mitochondrial content and shape, particularly in proliferating or differentiating stem cells and their progenies.Thereafter, we generate three independent hypotheses for the role of mitochondrial content and shape regulation towards understanding the distinction of these mitochondrial properties between proliferating and differentiated cells.We also propose approaches of testing these independent hypotheses with quantitative analyses of mitochondria.Finally, we discuss an example of a quantitative link between mitochondrial content and shape through cell size scaling, which we hypothesize to be existing during cell proliferation and differentiation of stem cells and their progenies.

Quantitative aspect of heterogeneity of mitochondrial content in proliferating versus differentiated cells
Mitochondria are double membrane bound organelles with at least one mt-DNA nucleoid.The mt-DNA codes for 13 protein coding genes that are transcribed and translated inside the mitochondrial matrix with the help of tRNAs and rRNAs coded by the mt-DNA [13].All the rest of the approximately 1100 mitochondrial proteins are coded by the nuclear DNA.Cells and tissue types vary in bioenergetics and metabolic abilities.Classically, mitochondria were studied primarily from aerobic differentiated tissues with higher mitochondrial content, like heart, liver and muscle.The tissue and cell type specific differences of mitochondria are largely maintained by distinct mitochondrial proteome (and phospho-proteome) and a varied mtDNA content between differentiated tissues [1,20,21].For example, the respiratory chain complex IV, which consumes oxygen to generate ATP (oxidative phosphorylation), exhibits tissue specific isoforms [20].
The stem cells, harbour only few mitochondria that are specialized to maintain stem cell functionality (table 2).Various cells respond to increased metabolic demand and stresses by increasing their mitochondrial content that is driven by master regulators of mitochondrial biogenesis, like PGC1-α, Myc and mTOR [22][23][24].To meet the demand of pathophysiologic situations, such as diabetes, heart failure or T-cell activation, mitochondria can also be remodelled during biogenesis [25][26][27].The regulation of amount and kind of mitochondria also involves the process of mitophagy that removes mitochondria in a selective or non-selective manner [28].Therefore, the make-up of the resultant mitochondrial content in any differentiated or proliferating cell is a function of mitochondrial biogenesis and mitophagy.
Mitochondria consist of all the major biological macromolecules, namely protein, lipid, DNA and RNA.Therefore, ideal analyses of mitochondrial content include assessment of the mitochondrial protein, lipid, mt-DNA (and RNA).The methods used are based on biochemical analyses, fluorescent and electron microscopy, polymerase chain reaction (PCR), flow cytometry, genomics and proteomics.These methods can quantitatively or semi-quantitatively measure parameters like mitochondrial protein levels, mitochondrial enzyme activities, mt-DNA levels, mitochondrial number, mitochondrial lipid content and gene expression.Employing a single method to estimate mitochondrial content could lead to errors, as reflected in the following examples.Use of mitotracker dyes in haematopoietic stem cells can be erroneous due to the ability of cells to actively extrude the dye [29].Also, mitochondrial content measured by transmission electron microscopy in human subjects did not correlate with mt-DNA content, while it strongly correlated with cardiolipin content and citrate synthase activity [30].A mass spectrometry-based approach for quantitative comparison of mitochondrial content across four mouse tissues used a novel parameter named 'mitochondrial enrichment factor' (MEF) [31].The authors, based on their observations, caution that levels or activity of no single mitochondrial protein (ex: citrate synthase) can be reflective of mitochondrial content as opposed to the standard practice.Also, mitochondrial number may not reflect mitochondrial content in cases where the majority of the mitochondrial content are in large hyperfused mitochondrial networks [32,33].Furthermore, an increase in mitochondrial content in response to mitochondrial stress may not always lead to increases in mitochondrial function [34].Therefore, accurate understanding of the functional significance of alteration of mitochondrial content is best possible with multiparametric analyses.Here, we have listed the different methods used to assess mitochondrial content and its royalsocietypublishing.org/journal/rsob Open Biol.14: 230279 regulation by mitochondrial biogenesis and/or mitophagy in various tissue/cell types, including stem cells (tables 1 and 2).It is noteworthy that the metrics obtained using different methodologies and detection platforms cannot be considered as absolute measures and thus cannot be compared between studies.
During the process of cell proliferation, the net mitochondrial content can only increase by twofold with the doubling of cellular mass, thus maintaining the scaling of mitochondrial and other cellular contents.Mitochondria have been demonstrated to partition in proportion with the cytosolic volume in yeast and randomly between daughters in symmetrically partitioning mammalian cells [90,91].Therefore, we reason that some active control of mitochondrial content is necessary during each round of cell proliferation.On the other hand, the cell differentiation process is associated with a dramatic increase in mitochondrial content [2,7,46,72,83,85].In some cases, the increase in mitochondrial content has been causatively linked with entry into differentiation by employing genetic/genomic approaches in vivo or in vitro models [43,70,[92][93][94][95]. Stem cells being a meaningful model to study differentiation, here we carefully surveyed the fold increase in mitochondrial content reported during stem cell differentiation.We consistently found differentiation of stem cells associates with a greater than twofold increase in mt-DNA copy number (that can exceed fivefold in some cases), across various studies on various cell lineages (table 2).Notably, comparison of other mitochondrial parameters measured did not reveal such a striking phenomenon in our survey.Consistently, measurement of certain mitochondrial parameters, excluding mt-DNA, has been found to be comparable between embryonic stem cells and their differentiated counterparts [81].Therefore, we hypothesize that while a self-renewing/proliferating cell maintains a strict control on twofold increase in the mt-DNA copy number, a greater than twofold increase in particularly mt-DNA copy number is sensed by the stem cell as a trigger for differentiation (hypothesis I in figure 1).Evidence for such a conceptualization can be obtained with quantitative measurement of mt-DNA content in stem cells during the event of cell cycle exit for entering differentiation (see proposed hypothesis testing section).

Quantitative aspects of heterogeneity of mitochondrial shape and dynamics in proliferating versus differentiated cells
Subjective evaluation of mitochondrial shape within cells has revealed a wide spectrum, which can be discontinuous (discrete shapes) or continuous (different mitochondrial length, number or size).Examples of subjective descriptions of mitochondrial shape are clustered, aggregated, doughnut shaped, networked, tubular, hyperfused, net-like and fragmented [3,96,97].An expanding body of literature also demonstrates that various pathophysiological stimuli alter mitochondrial shape in a given cell type [17,98,99].Mitochondrial shape in any given cell is maintained by a balance in the dynamic opposing processes of mitochondrial fission and fusion that are collectively referred to as mitochondrial dynamics [97][98][99].Mitochondrial fission refers to the process where a larger mitochondrion undergoes fission of their inner and outer membranes to form smaller mitochondria.Mitochondrial fission is driven by dynamin-related protein 1 (Drp1) that is recruited to the mitochondrial surface from the cytosol by bona fide mitochondrial proteins like mitochondrial fission factor 1 (MFF1) or fission protein 1 (Fis1).On the other hand, mitochondrial fusion refers to the process where two smaller mitochondria fuse both their membranes to form a larger mitochondrion.Transient fusion events happening between two mitochondria allow exchange of contents between them, without forming a larger mitochondrion.The outer mitochondrial fusion is driven by mitofusins (Mfn1/2) while the inner membrane fusion is primarily driven by optic atrophy 1 (Opa1).Influence of cytoskeletal elements and the endoplasmic reticulum on mitochondrial fission and fusion processes further reveals the complexity of how mitochondrial dynamics maintain the morphometric features of a given mitochondrial shape [100,101].
Quantitative understanding of mitochondrial shape along its wide morphometric spectrum is crucial for obtaining deeper insight into the structure-function relationship of mitochondria and how that impacts various cellular processes including differentiation of stem cells [97][98][99].Analyses of mitochondrial morphometry obviously involves electron and fluorescence microscopy-based visualization and various image analyses tools.Tools to quantify mitochondrial morphometric parameters from micrographs, primarily of cells and tissues that are amenable to fluorescence microscopy, include multiple Image J plugins [102], MitoHacker [103] (for high-throughput two-dimensional analyses), MitoGraph (for high-resolution three-dimensional analyses) [104] and various customized algorithms [32,105,106].A mitochondrial morphological complexity index of mouse hippocampus has been recently reported using a three-dimensional approach employing serial block face scanning electron microscopy [9].MitoGraph output has been used in the MitoSinCe 2 method to design quantitative metrics for assessing the contribution of mitochondrial fission/fusion on their structure-function in single cells [107].An automated approach, named Mitometer, has been reported for identifying and quantifying mitochondrial fission and apparent fusion events by tracking dynamic mitochondria in cells [108].Assessment of definitive fusion events of mitochondrial inner and outer membranes has been achieved by employing photoconvertible fluorescent probes in live cell pulse chase assays [107,109].
A clear distinction of mitochondrial shape between stem/ progenitor cells and their differentiated counterparts has been reported in various lineages [96,[110][111][112][113][114] (table 3).The majority of the studies on pluripotent stem cells (ESCs and iPSCs) have reported small punctate mitochondria with less matured cristae and bioenergetic functionality in the stem cells and elongated mitochondria in their differentiated counterparts [87,113,117,120,124,129].On the other hand, elongated and fused mitochondria have been reported in adult stem cells of various lineages [8,96,112,124,129,134,[137][138][139][140], with some lineages maintaining punctate mitochondria [114].Differentiation in stem cells has been shown to be supported by either mitochondrial fission or fusion, as investigated in different cell lineages [8,112,129,134,[137][138][139][140].The majority of such studies investigated the impact of mitochondrial shape at the extremes of the morphometric spectrum, largely leaving out the shapes in between the extremes.Precise quantification of mitochondrial network length revealed that maintenance of royalsocietypublishing.org/journal/rsob Open Biol.14: 230279 royalsocietypublishing.org/journal/rsob Open Biol.14: 230279 smaller network size (comprising of ≤ 40% of the total mitochondrial network size) supports stem cell gene expression profile in keratinocyte lineage, while almost complete fusion of mitochondrial network (hyperfusion) prevents it [128].The smaller mitochondrial network size is reminiscent of the theoretical conceptualization of a quantitively defined 'meso-fused' royalsocietypublishing.org/journal/rsob Open Biol.14: 230279 mitochondrial structure, where the active fission rate dominates to maintain smaller mitochondrial network size [141].Dramatic alteration of mitochondrial shape happens both during the proliferating cycle of cells and in their differentiation process towards regulating such physiology [96,110,113,114].In the light that mitochondrial physiology is distinct between proliferating and differentiated cells [142,143], the regulation and role of mitochondrial fission and fusion dynamics can also be potentially different between them.For instance, the estimated rate of mitochondrial fission and fusion dynamics in differentiated myocytes is of the order of days as opposed to seconds as observed in in vitro culture systems [144,145].The higher rate of mitochondrial dynamics observed in differentiated cell line models or ex-vivo tissue models could be confounded by their proliferating status or isolation-stress induced change in mitochondrial shape [146].Indeed, the rate of mitochondrial dynamics increases as quiescent mouse embryonic fibroblasts (alike quiescent differentiated cells) are induced to enter cell proliferation (unpublished results).Predictive theoretical considerations have revealed that mitochondrial fission and fusion rates can influence mitochondrial shape and dynamics independently [141].Steady state mitochondrial shape (as observed in snapshot micrographs) can be determined by the ratio of fusion and fission rates, i.e. the number of mitochondrial fission or fusion events per unit time (as quantified from time lapse micrographs).In scenario 1, a cell with two fusion events/mito-element/minute and one fission event/ mito-element/minute can potentially achieve and maintain a moderately fused network with fast mitochondrial dynamics.In scenario 2, a cell with two fusion events/mito-element/ day and one fission event/mito-element/day would have identical steady state mitochondrial shape as in scenario 1 but exhibit much slower mitochondrial dynamics.We hypothesize that if scenario 1 represents a proliferating cell, scenario 2 with lower rates of mitochondrial fission and fusion events may represent the differentiated counterpart of that cell (hypothesis II in figure 1).Evidence for such a conceptualization can be obtained by measurement of absolute rates of mitochondrial fission and fusion events with advanced microscopy and image analyses techniques in proliferating cells and their differentiated counterparts (see proposed hypothesis testing section).

Quantitative analyses reveal link between mitochondrial content and shape in cell size regulation
The regulatory balances for mitochondrial shape (fission and fusion) and of mitochondrial content (biogenesis and mitophagy) have been proposed to be integrated through a mitochondrial life cycle [18].Here, it is important to make the conceptual distinction between mitochondrial fission and division.Only mitochondrial division, and not fission, increases overall mitochondrial content when not balanced out by mitophagic clearance.On the other hand, fusion of two smaller mitochondria would not amount to mitochondrial biogenesis without a concomitant increase in the mass of the resultant fused mitochondria from that of the sum of the individual mitochondria.In the mitochondrial life cycle, mitochondrial fission and fusion counter each other, which is quantitatively captured in the inverse relationship of the metric measuring contribution of fission and fusion on mitochondrial shape [107].The inverse relationship of mitochondrial fission and fusion metrics weakens below a certain level of the fusion metric.It remains to be quantitatively examined if and how modulation of mitochondrial biogenesis and/or mitophagic clearance may impact the aforementioned inverse relationship.However, mitophagic clearance is thought to operate on punctate mitochondria, and maintenance of mitochondrial tubules can prevent that [18,28].Mitochondrial fission towards the end of a mitochondrial tubule can also be associated with mitophagic machineries in proliferating cells, whereas mitochondrial fission in the middle of a tubular mitochondria is associated with replicating mt-DNA [147].However, replicating mt-DNA cannot be equated to mitochondrial biogenesis that results in an overall increase in mitochondrial content.Genetic manipulation of mitochondrial fission or fusion machineries can dramatically impact mitochondrial content, biogenesis and mitophagy [7,144,145,148].Extensive hyperfusion by greater than 90% reduction of the mitochondrial fission protein, Drp1, elevates mitochondrial number and gene expression of mitochondrial proteins and prevents stem cell maintenance in skin keratinocyte lineage [128].Importantly, such a compensatory effect is not observed with fine-tuned repression of Drp1 that sustains stem cell gene expression in the same lineage [128].Associated increases in mitochondrial fission and mitochondrial content have been causatively linked with the process of stem cell differentiation [45,71,110,113,114].Excessive mitochondrial fission causes a bioenergetic crisis to potentially turn on the compensatory mechanism of mitochondrial biogenesis and push the cells out of the cell cycle and towards differentiation [98,144].
In general, differentiated cells are larger than their lineage specific stem cells [19].Increase in cell size needs an overall boost in gene transcription and translation supported by increased anabolic activities, where biosynthetic and metabolic activities are scaled with cell size [149].Overall, the gene transcription rate and protein levels are expected to be higher in proliferating cells with higher mitochondrial Figure 1.(Overleaf.)Three independent hypotheses are postulated on mitochondrial shape and content during cell differentiation, as proliferating cells undergo cell cycle exit, active differentiation and post differentiation.Hypothesis I, on the dependence of differentiation on mtDNA content, states: while a self-renewing/proliferating cell maintains strict control on a twofold increase in the mt-DNA copy number, a greater than twofold increase in mt-DNA copy number is sensed by the stem cell as a trigger for differentiation.Hypothesis II, on the differences of rates of mitochondrial fission-fusion between differentiated and proliferating counterparts, states: if scenario 1 represents a proliferating cell, scenario 2 with lower rates of mitochondrial fission and fusion events may represent the differentiated counterpart of that cell.Hypothesis III, on allometric scaling of mitochondria with cell size during differentiation, states: the reported quantitative allometric scaling of mitochondrial content/functionality with cell size, through alteration of mitochondrial shape, is established during differentiation of stem cells.The panel at the bottom indicates that the interaction of three independently hypothesized events (H-I, H-II, H-III) can potentially happen during any or all of the steps of cell cycle exit, active differentiation or post-differentiation.
royalsocietypublishing.org/journal/rsob Open Biol.14: 230279  [68,150].The Largen gene, isolated in a screen to identify cell size regulatory genes, substantially increases mitochondrial content and boosts mitochondrial respiratory function by specifically augmenting the translation of mRNAs encoding mitochondrial proteins [151].
Similarly, the scaling of mitochondrial network size with cell size has been found to be linear in the growing yeast bud [38].However, in a genetically engineered non-proliferating liver cell model that can grow in size, gene transcription for mitochondrial proteins was found to reduce in larger cells [152].This observation on liver cells, which have higher ploidy than diploid cells, is consistent with the observations in other polyploid models [153][154][155].Moreover, the quantitative allometric relationship of mitochondrial functionality (as measured by potentiometric dye incorporation) and cell size appears to be nonlinear, even in diploid proliferating cell models.There, the mitochondrial function is maximum in cells of intermediate size, beyond which it reduces with an increase in cell size [36].It has been proposed that mitochondrial fission and fusion abilities change mitochondrial shape to compensate for reduced mitochondrial functionality, thus allowing effective mitochondrial function in larger cells.Such a conclusion is based on the result that causing mitochondria to hyperfuse by Drp1 repression boosts mitochondrial function only in larger cells within diploid proliferating populations [36,156].It has also been proposed from quantitative theoretical analyses that other factors like cell cycle can also potentially influence the scaling of mitochondria content/function and cell size [157].royalsocietypublishing.org/journal/rsob Open Biol.14: 230279 The increase in mitochondrial content and cellular mass is molecularly coupled by the mTOR pathway that sustains the cellular anabolic activities related to cell growth [24] and supports stem cell differentiation of various lineages [158].The energy demanding process of protein translation, regulated by the mTOR pathway, has been proposed to be dependent on mitochondrial energy [159,160], while the mTOR pathway also contributes to the maintenance of mitochondrial content [161].Therefore, feedback between mitochondrial content and mTOR-driven protein translation has been proposed, where the quantitative aspects of the feedback loop remain an open question.Such a feedback loop could be potentially at play between mTOR and the mitochondrial fission protein Drp1 in the following way.Repression of ribosomal activity with mTOR inhibition represses Drp1 driven mitochondrial fission through the fission regulator MTFP1 [162].Whereas, repression of Drp1 has been noted to be causatively or correlatively linked to elevated expression of ribosomal genes in proliferating cells, including stem cells [128,133,163].However, in differentiated cardiac and muscle tissues, Drp1 repression reduces the expression of ribosomal genes and protein synthesis [164,165].This difference in the impact of mitochondrial fission adds to the distinction in mitochondrial behaviour between proliferating and differentiated cells.The two other regulatory circuitries for cell size control, namely the Hippo kinase pathway and Myc, also have been shown to crosstalk with mitochondrial shape and/or mitochondrial content [22,[166][167][168].Self-renewing stem cells are usually smaller in size than their differentiated counterparts in the majority of the lineages, indicating the increase in cell size happens along the time line of differentiation [19].Therefore, we hypothesize that the reported quantitative allometric scaling of mitochondrial content/functionality with cell size, through alteration of mitochondrial shape, is established during differentiation of stem cells (hypothesis III in figure 1).

Proposed hypotheses testing
Studying the significance of maintaining and modulating a particular mitochondrial shape in a given cell is an active field of research and has been covered in several reviews [98,99,169].The standard approach generally includes three steps: (a) defining the mitochondrial shape; (b) establishing the correlation of mitochondrial shape with levels/activity of mitochondrial fission and fusion proteins; (c) studying the mechanism of impact of genetic or pharmacological manipulation of the relevant mitochondrial fission or fusion proteins on mitochondrial structure-function and the relevant cell physiology.The majority of the studies on the elucidation of the significance on mitochondrial shape change in stem cell differentiation compare mitochondrial shapes between the stem cells (start point) and their differentiated counterparts (end point).Therefore, some key questions remain open about the timeline of various events during the course of stem cell differentiation.For example, (a) when and how does the mitochondrial shape change happen; (b) when and how the mitochondrial fission and fusion processes are connected to other concomitant mitochondrial changes (ex: increase in mt-DNA copy number) and cellular changes (ex: increase in cell size); (c) the exact functional significance of such mitochondrial shape change.The altered mitochondrial shape and content observed in differentiated cells, their potential interdependence and connection to cell size may be achieved during cell cycle exit, during the process of differentiation or postdifferentiation, or in any possible combinations during the course of differentiation (figure 1).Establishment of such a timeline would then allow investigation of the functional significance of such alterations in stem cell differentiation using conditional gene manipulation strategies.The three independent hypotheses laid out in the previous three sections can be tested in a relevant stem cell model along the time course of cell cycle exit, active differentiation and after completion of differentiation.The relevant mitochondrial properties, namely mitochondrial shape metrics, mitochondrial fission-fusion rates, mitochondrial content metrics, and cell size metrics, can be measured in single cells with quantitative approaches discussed in the relevant sections.Thereafter, quantitative correlation and mechanistic causation can be established between parameters in the differentiation time line.
In this review, we have covered relevant findings from mammalian cells as well as yeast models, where the latter has proved to be extremely important in understanding fundamental and general concepts of mitochondrial shape.Therefore, the concepts discussed here may also apply to the fascinating diversity of mitochondrial shape and behaviour existing in other taxa like plants, Chlamydomonas, Planaria, and others.

Table 1 .
Measurement of mitochondrial content in differentiated cells.

Table 2 .
Measurement of mitochondrial content in stem cells.

Table 3 .
Mitochondrial shape in stem cells.